1. Field of the Invention
This invention relates to particle measuring and counting apparatus, as incorporated into flow cytometers. The invention is particularly well suited to the measurement of small particles such as microspheres (typically 10 nm to 10 um diameter), cells (eg blood cells or bacteria), parts of cells (eg nuclei), or viruses.
2. Description of Related Art
A flow cytometer counts, measures and discriminates particles in a liquid by their optical properties as they flow through a beam of illuminating radiation one particle at a time. The radiation source is typically a laser and the machine is normally used to count and identify particles at rates up to roughly 100000 particles per second.
The sample is normally prepared by labelling it with one or more fluorescent markers. Each fluorescent marker emits light of a characteristic wavelength range (colour) when it is excited by the laser light. The fluorescent marker may be present in the particle in a quantity roughly proportional to a substance in the particle (for example the particle's DNA content), and therefore the fluorescence signals may indicate certain features of the particle.
Flow cytometers are typically equipped with several optical detectors. Independent optical detectors may be fitted to measure light scattered at a variety of angle ranges, typically described as Small Angle Light Scatter (roughly 1 to 15 degrees, “SALS”), Medium Angle Light Scatter (roughly 15 to 60 degrees, “MALS”) and Large Angle Light Scatter (typically greater than 60 degrees, “LALS”), and optical detectors to measure several different colours of fluorescence. The scatter angles are chosen to optimise the resolution between different populations of particle.
The sample liquid is hydro-dynamically focused into a narrow sample core. This may be done by a sheath fluid as it flows into the flow cell channel. Particles in the sample liquid thus pass through a point of detection in the flow cell channel one at a time and are measured individually. A light source (typically a laser) is focussed at the point of detection in the flow channel and this light is scattered by particles travelling through the flow cell. If labelled with a fluorescent marker, the particles will also emit light by fluorescence.
The scattered and fluoresced light is converted to an electrical pulse by optical detectors (typically photomultipliers), and the size and shape of these pulses is recorded by computer.
The pulse measurements are typically plotted on histogram graphs such that particles with different characteristics form distinct populations on the histograms.
It is well known that flow cytometry offers a means for counting and discriminating mammalian cells and some bacteria. However, many bacteria, archaea and viruses are too small for conventional flow cytometers to measure precisely, particularly by light scattering.
To measure differences between small particles such as bacteria, a relatively high sensitivity flow cytometer is required. It is well known that the sensitivity of a well designed flow cytometer is limited by its optical performance. The optical sensitivity may be optimised by collecting as much signal as possible from the particle, and by eliminating as much background light (noise) as possible.